Lipopolysaccharide (LPS), commonly used to mimic bacterial infection without using live bacteria, was injected into roosters from three genetically diverse lines and gene expression was measured at two time points post-injection. TLR4, MD-2, and MyD88 are three genes that are responsible for initiating the major immune pathway that responds to LPS. Results showed significant differences in gene expression at different times post-stimulation for both MyD88 and TLR4. The three chicken lines had different expression levels of TLR4 in response to LPS. This shows that there are genetic differences in this immune pathway. Further studies are needed, but it may be possible to use TLR4 expression information in the selection process. Overall, bacterial infections are a serious threat to chickens and a better understanding of this pathway will lead to beneficial, applied approaches in the poultry industry.